Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



1.Research Institution@The University of Tokyo
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2.Research Area@Life Sciences
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3.Research Field@Structure and Functional Control Mechanism of Biomolecules (Structural Biology and Functional Molecules)
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4.Term of Project@FY1996`FY2000
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5.Project Number@96L00505
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6.Title of Project@Structural Change of Signal-transducing Proteins Induced by Ligand Binding: Its Significance in Signal Generation

7.Projetct Leader
Name Institution,Department Title of Position
Toshiaki, KATADA The University of Tokyo, Grad. Sch. of Pharmaceut. Sci., Professor

8.Core Members

Names Institution,Department Title of Position
Yoshinori, SATOW The University of Tokyo, Grad. Sch. of Pharmaceut. Sci., Professor
Ichio, SHIMADA The University of Tokyo, Grad. Sch. of Pharmaceut. Sci., Professor
Hiroyuki, ARAI The University of Tokyo, Grad. Sch. of Pharmaceut. Sci., Professor

9.Cooperating Researchers

Names Institution,Department Title of Position
Hiroshi, NISHINA The University of Tokyo, Grad. Sch. of Pharmaceut. Sci., Associate Professor
Shin-ichi, HOSHINO The University of Tokyo, Grad. Sch. of Pharmaceut. Sci., Research fellow
Kenji, KONTANI The University of Tokyo, Grad. Sch. of Pharmaceut. Sci., Research fellow

10.Summary of Research Results

In the present study, we analyzed the structures and functions of various proteins involved in signal-transduction systems. Three-dimensional structural analysis by X-ray crystallography and NMR was performed especially for the signal-transduction processes of ligand-protein and protein-protein interaction. We obtained the following new findings. 1. A novel G protein GSPT was identified as the eukaryotic polypeptide releasing factor eRF3. GSPT/eRF3 functioned not only in translation termination but also in mRNA degradation. The N domain of eRF3 associated with PABP to unmask the poly(A) tail of mRNAs for their degradation. Novel molecules were identified as binding proteins for the small GTPase Rab5, which was also capable of binding to the p110/p85 subtype of phosphoinositide 3-OH kinases. The Rab5-binding proteins were transferred into nucleus at G2-M phase in a manner dependent on GTP-bound Rab5. 2. Three-dimensional structural studies by X-ray crystallography were carried on the liganded proteins of glutathione S-transferase, antibody Fab and Fv fragments, human renal dipeptidase, Fabry disease enzyme of human -galactosidase. Large-scale expression systems were developed for the structural studies of human interleukin-6 receptor and 3-adregenic receptor. 3. By NMR analysis, we showed that stoichiometry of the Fc-sFcRII interactions 1:1. An extensive mapping of the sFcRII-binding site of Fc, which was performed by use of stable-isotope-assisted NMR spectroscopy, led us to conclude that sFcRII binds to the lower hinge and its spatial proximity of the IgG-Fc. We also developed a stable isotope-labeling method for glycans attached to glycoproteins and a method used for the identification of the interfaces of larger protein-protein complexes in solution. 4. Three-dimensional structural analysis by X-ray crystallography was performed on the subunit of PAF acetylhyrolase. The subunit was structurally quite similar to the trimeric G protein subunit. The LIS1/ subunit appeared to function in nuclear migration by interacting with multiple intracellular proteins.

11.Key Words

(1)G proteinA(2)Translation terminationA(3)mRNA decay
(4)GlycoproteinA(5)AntibodyA(6)Recepror
(7)PAF acetylhydrolaseA(8)X-ray crystallographyA(9)NMR

12.References

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S. Hoshino, et al. The eukaryotic polypeptide chain releasing factor(eRF3/GSPT)carrying the translation termination signal to the 3'-Poly(A) tail of mRNA: Direct association of eRF3/GSPT--
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N. Hosoda, et al. Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycans.
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Eur. J. Biochem. 265 1999 763-770

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S. Hoshino, et al. Novel function of the Eukaryotic Polypeptide-chain Releasing Factor 3 (eRF3/GSPT)in the Mechanism of mRNA Degradation.
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Biochemistry(Moscow) 64 1999 1367-1372

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T. Katada, et al. Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.
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Chemistry and Physics of Lipids 98 1999 79-86

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M. Nishida, et al. Gi and Go are target proteins of reactive oxygen species.
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Nature 408 2000 492-495

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T. Katada, et al. Enzymic and signal transduction properties of CD38/NADase and PC-1/phos-phodiesterase.
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Chem. Immunol. 75 2000 60-78

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M. Hara-Yokoyama, et al. Complex Gangliosides as Cell Surface Inhibitors for the Ecto-NAD+ Glycohydrolase of CD38.
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Biochemistry 40 2001 888-895

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T. Wada, et al. Impaired synergistic activation of stress-activated protein kinase SAPK/JNK in ES cells lacking SEK1/MKK4.
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J. Biol. Chem. 276 2001 in press

Author Title of Article
H. Yokoyama, R. Mizutani, Y. Satow, Y. Komatsu, E. Ohtsuka, and O. Nikaido Crystal structure of the 64M-2 antibody Fab fragment complexed with a DNA dT(6-4)T photoproduct formed by ultraviolet radiation
Journal Volume Year Pages Concerned
J. Mol. Biol. 299 2000 711-723

Author Title of Article
T. Shiba, S. Harada, H. Sugawara, H. Naitow, Y. Kai, and Y. Satow Crystallization and Preliminary X-ray Analysis of a Bacterial Lysozyme Produced by Streptomyces globiporus
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Acta Cryst. D56 2000 1462-1463

Author Title of Article
H. Yokoyama, R. Mizutani, and Y. Satow Complex Formation of Double-Stranded DNA(6-4) Photoproducts and Anti-(6-4) Photoproduct Antibody Fab
Journal Volume Year Pages Concerned
Nucleic Acids Symposium Series 44 2000 151-152

Author Title of Article
T. Meguro, T. Kashiwagi, and Y. Satow Crystal Structure of the Low-Humidity Form of Aspartame Sweether
Journal Volume Year Pages Concerned
J. Peptide Research 56 2000 97-104

Author Title of Article
M. Nishida, S. Harada, S. Noguchi, Y, Satow, H. Inoue, and K. Takahashi Three-Dimensional Structure of Glutathione S-Transferase from Escherichia coli
Journal Volume Year Pages Concerned
Clinical Chemistry and Enzymology Communications 8 1999 271-276

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H. Yokoyama, R. Mizutani, Y. Satoww, Y. Komatsu, E. Ohtsuka, and O. Nikaido Crystal Structure of the 64M-2 and 64M-3 Antibody Fabs Complexed with DNA(6-4) Photoproducts
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Nucleic Acids Symposium Series 42 1999 267-268

Author Title of Article
R. Mizutani, Y. Satow, Y. Ohya, M. Kawasaki, and Y. Anraku Crystal Structure of the VMA1-Derived Endonuclease Bearing an N-Terminal Propeptide
Journal Volume Year Pages Concerned
Acta Cryst. A55 1999 280

Author Title of Article
S. Miyamoto, R. Mizutani, Y. Satow, M. Kawasaki, Y. Ohya, and Y. Anraku Recognition and Cleavage of Double-Stranded DNA by Yeast VMA1-Derived Endonuclease
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Nucleic Acids Symposium Series 42 1999 197-198

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H. Yokoyama, R.Mizutani, Y. Satow, Y. Komatsu, E. Ohtsuka, and O. Nikaido Crystal Structures of the 64M-2 Antibody Fab Fargment and Its Complex with DNA(6-4) Photoproducts
Journal Volume Year Pages Concerned
Acta Cryst. A55 1999 307

Author Title of Article
M. Nishida, S. Harada, S. noguchi, Y. Satow, H. Inoue, and K. Takahashi Three-Dimensional Structure of Escherichia coli Glutathione S-Transferase Complexed with Glutathione Sulfonate:Catalytic Roles of Cys 10 and His 106
Journal Volume Year Pages Concerned
J. Mol. Biol. 281 1999 135-147

Author Title of Article
S. Noguchi, K. Miyawaki, and Y. Satow Succinimide and Isoaspartate Residues in the Crystal Structures of Hen Egg-White Lysozyme Complexed with Tri-N-Acetylchitotriose
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J. Mol.Biol. 278 1998 231-238

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N. Noda, S. Noguchi, and Y. Satow Crystal Structures of Nucleic Acid Complexes of Ribonuclease U2
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Chiaki Nishimura Folding topologies of Human Interleukin-6 and its Mutants as Studied by NMR Spectroscopy
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Biochemistry 35 1996 273-281

Author Title of Article
Satoko Akashi Characterization of Mouse Switch Variant Antibodies by Matrix-Assisted LaserDesorption Ionization Mass Spectroscopy and Electrospray Ionization Ma
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J. Am. Soc. Mass. Spectrom. 7 1996 707-721

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Jae Yoon Leem Purification of Caracterization of N-b-anayl-5-S-gltathionyl-3, 4dihydroxy phenyllanlanine, a Novel Antibacterial Substance of Sarcophaga peregrin(fresh
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J. Biol. Chem. 271 1996 13573-13577

Author Title of Article
Hiroyuki Ohsuga The Carbohydrate Moiety of the Bermuda Grass Antigen BG60
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Author Title of Article
Ryuta Mizutani Crystallization and crystallographic studies on Fab fragments of anti-nitrophenol antibodies from primary and secondary immune responses
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J. Crystal Growth 168 1996 253-259

Author Title of Article
Junichi Enokizone NMR analysis of the interaction between protein L and Ig light chains
Journal Volume Year Pages Concerned
J. Mol. Biol. 270 1997 8-13

Author Title of Article
Tsu-An Hsu Differential N-Glycan Patterns of Secreted and Intercellular IgG Produced in Trchoplusia ni Cells
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J. Biol. Chem. 272 1997 9062-9070

Author Title of Article
Daisaku Sato Stereoselective Reactions. 26. Solution Structures of a Chiral Bidentate Lithium Amide in Relation to the Solvent-dependent Enantioselectivites in D
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Tetrahedron 53 1997 7191-7200

Author Title of Article
Satoko Akashi Structural Characterization of Mouse Monoclonal Antibody 13-1 against a Porphyrin derivative: Identification od a Disulfide Bond in CDR-H3 of Mab 13-
Journal Volume Year Pages Concerned
Biochem. Biophys. Res. Commun. 240 1997 566-572

Author Title of Article
Hiroaki Gouda NMR Study on the Interaction between the B Domain of Staphylococcal Protein A and the Fc Portion of Immunoglobulin G
Journal Volume Year Pages Concerned
Biochemistry 37 1998 129-136

Author Title of Article
Hiroyuki Kobayashi Specificities and Rate of Binding of Anti-(6-4) Photoproduct Antibody Fragment to Synthetic Thymine Photoproducts.
Journal Volume Year Pages Concerned
J. Biochem(Tokyo) 123 1998 182-188

Author Title of Article
Kazuo Hirayama Complete and Rapid peptide-and glyco-mapping of Mouse Monoclonal Antbody By LC/MS/MS Using Ion Trap Mass Spectroscopy
Journal Volume Year Pages Concerned
Analytical Chemistry 70 1998 2718-2725

Author Title of Article
Yoshiki Yamaguchi Development of a Systematic Method for13 C Labeling of LgG Carbohydrate Chains for NMR Study
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Journal of Biomolecular NMR 12 1998 385-394

Author Title of Article
Takuya Torizawa 31P NMR Study of the Interactions between Oligodeoxynucleotides Containing (6-4) Photoproduct and Fab Fragments of Monoclonal Antibodies Specific for
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FEBS Lett. 429 1998 157-161

Author Title of Article
Noriko Takahashi N-glycan structure of a recombinant mouse soluble Fc receptor II
Journal Volume Year Pages Concerned
Glycocojugate Journal 15 1998 905-914

Author Title of Article
Kuniko Akama Transition Protein I from Boar Spermatid Nuclei Having DNA-melting Activity Is A Dimeric Protein
Journal Volume Year Pages Concerned
Biochem. Mol. Biol. Int. 44 1998 315-323

Author Title of Article
Nobuhisa Shimada Determination of Protonation, Deprotonation Forms and Tautomeric States of Histidine Residues in Large Proteins by Using Nitrogen-Carbon J Couplings
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J. Am. Chem. Soc. 120 1998 10988-10989

Author Title of Article
Kazuo Hirayama Convenient Peptide Mapping of Immunoglobulin G2b and Differentiation between Leucine and Isoleucine Residues by Mass Spectrometory Using 2H-
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J. Mass Spectrom. Soc. Jpn. 46 1998 83-89

Author Title of Article
Hiroyuki Kobayashi Probing the Interaction between a High-Affinity Single-Chain Fv and a Pyrimidine(6-4) Pyrimidone Photodimer by Site-Directed Mutagenesis
Journal Volume Year Pages Concerned
Biochemistry 38 1999 532-539

Author Title of Article
Takuya Torizawa Conformational Multiplicity of the Antibody Combining Site of a Monoclonal Antibody Specific for a (6-4) Photoproduct
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J. Mol. Biol. 290 1999 731-740

Author Title of Article
Shunro Kawaminami A Multinuclear NMR Study of the Active Site of an Endoglucanase from a Strain of Bacillus: Use of Trp Residues as Structural Probes
Journal Volume Year Pages Concerned
J. Mol. Chem. 274 1999 19823-19828

Author Title of Article
Masayoshi Nakasako The pH-dependent structural variation of complementarily-determining region H3 and the association mode of variable domains in Fv fragment
Journal Volume Year Pages Concerned
J. Mol. Biol 291 1999 117-134

Author Title of Article
Yoshiki Yamaguchi Development of a Systematic Method for13 C Labeling of IgG Carbohydrate Chains for NMR Study
Journal Volume Year Pages Concerned
Glycocojugate J. 16 1999 8

Author Title of Article
Noriko Takahashi N-glycan structure of a recombinant mouse soluble Fc receptor II
Journal Volume Year Pages Concerned
Glycocojugate J. 16 1999 114

Author Title of Article
Katsuyoshi Masuda Post-translational modifications of immunogolobulin G: A mouse IgG variant That lacks the entire CH1 domain
Journal Volume Year Pages Concerned
Molecular Immunology 36 1999 993-1003

Author Title of Article
Hideo Takahashi Solution Structure of Hanatoxin 1, Gating Modifier of Drk1 Potassium Channel
Journal Volume Year Pages Concerned
Peptide Science @ 1999 83-86

Author Title of Article
Koh Takeuchi Three-dimensional Solution Structure of Calcium Channel Activator:: Imperatoxin A Determined by NMR Spectroscopy
Journal Volume Year Pages Concerned
Peptide Science @ 1999 83-86

Author Title of Article
Sato K Chemical synthesis and properties of (6-4) photoproduct and its analogs
Journal Volume Year Pages Concerned
Nucleic Acids Symposium Series. 42 1999 37-38

Author Title of Article
Hideo Takahashi A Novel NMR Method for the Determination of the Interface of Large Protein-protein Complexes
Journal Volume Year Pages Concerned
Nature Struct. Biol 7 2000 220-223

Author Title of Article
Koichi Kato Structural Basis of the Interaction between IgG and Fc Receptors
Journal Volume Year Pages Concerned
J. Mol. Biol. 295 2000 213-224

Author Title of Article
Kazuyasu Shindo Backbone 1H, 13C, and15N resonance assignments of the anti-dansyl antibody Fv fragment
Journal Volume Year Pages Concerned
J. Biomol. NMR 17 2000 357-358

Author Title of Article
Ichio Shimada NMR Analysis of Interactions Between Bio-Macromolecules
Journal Volume Year Pages Concerned
Real-Time Analysis of Biomolecular Interactions(K. Nagata and H. Handa, ed. ) @ 2000 223-231

Author Title of Article
Katsuyoshi Masuda Pairing of oligosaccharides in the Fc region of immunoglobulin G
Journal Volume Year Pages Concerned
FEBS Lett. 473 2000 349-357

Author Title of Article
Yoshiki Yamaguchi 1Hand 13C NMR assignments for the glycans in glycoproteins by using H C-labeled glucose as a metabolic precursor
Journal Volume Year Pages Concerned
J. Biomol. NMR 18 2000 357-360

Author Title of Article
Noriko Takahashi N-Glycan structure of murine hippocampus serine protease, neuropsin, produced in Trichoplusia ni cells
Journal Volume Year Pages Concerned
Glycoconjygate J 16 2000 405-414

Author Title of Article
Takuya Torizawa DNA-binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane thymine dimer
Journal Volume Year Pages Concerned
Nucleic Acid Res. 28 2000 944-951

Author Title of Article
Kousuke Sato Efficient chemical synthesis of a pyrimidine(6-4) pyrimidone photoproduct analog and its properties
Journal Volume Year Pages Concerned
Tetrahedron Lett. 41 2000 2175-2179

Author Title of Article
Hideo Takahashi Solution structure of hanatoxinl, a gating modifier of voltage-dependent K+channels:common surface features of gating modifier toxins
Journal Volume Year Pages Concerned
J. Mol. Biol. 297 2000 771-780

Author Title of Article
Nothwang, H. G. et al. Functional hemizygosity of PAFAH1B3 due to a PAFAH1B3-CLK2 fusion gene in a female with mental retardation, ataxia and atrophy of the brain
Journal Volume Year Pages Concerned
Hum. Mol. Gene. In press @ @

Author Title of Article
Jung-Hyun Min et al. Platelet-Activating Factor Acetylhydrolases: Broad Substrate Specificity and Lipoprotein Binding Does Not Modulate the Catalytic Properties of the Plasma Enzyme
Journal Volume Year Pages Concerned
Biochemistry In press @ @

Author Title of Article
Yasuda, K. et al. Platelet-Activating Factor Acetylhydrolase Isoforms I and II in Human Uterus. Comparisons with Pregnant Uterus and Myoma
Journal Volume Year Pages Concerned
Biol. Reprod. 64 2001 339-344

Author Title of Article
Bae, K. A. et al Platelet-activating Factor(PAF)-Dependent Transacetylase and Its Relationship with PAF Accetylhydrolases.
Journal Volume Year Pages Concerned
J. Biol. Chem. 275 2000 26704-26709

Author Title of Article
Kitagawa, M.. et al Direct association of LIS1, the lissencephaly gene product, with a mammalian homologue of a fungal nuclear distribution protein, rNUDE.
Journal Volume Year Pages Concerned
FEBS Letter 479 2000 57-62

Author Title of Article
Manya, H. et al Biochemical characterization of various catalytic complexes of the brain platelet-activating factor acetylhydrolase.
Journal Volume Year Pages Concerned
J. Biol. Chem. 274 1999 31827-31832

Author Title of Article
Manya, H. et al Biochemical characterization of various catalytic complexes of the brain platelet-activating factor acetylhydrolase.
Journal Volume Year Pages Concerned
J. Biol. Chem. 273 1998 18567-18572

Author Title of Article
Wei, Y. et al Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: streptomyces exfoliatus lipase at 1.9A resolution.
Journal Volume Year Pages Concerned
Structure 6 1998 511-519

Author Title of Article
Matsuzawa, A. et al Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.
Journal Volume Year Pages Concerned
J. Biol. Chem. 272 1997 32315-32320

Author Title of Article
Ho, Y. S. et al Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer.
Journal Volume Year Pages Concerned
Nature 385 1997 89-93

Author Title of Article
Hattori, K. et al cDNA cloning and expression of intracellular platelet-activating factor(PAF) acetylhydrolase II.
Journal Volume Year Pages Concerned
J. Biol. Chem. 271 1996 33032-33038

Author Title of Article
U. Albrecht. et al Platelet-activating factor acetylhydrolase expression and activity suggest a link between neuroral migration and platelet-activating factor.
Journal Volume Year Pages Concerned
Dev. Biol. 180 1996 579-593


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